It is now possible to introduce defined mutations into the DEN4 cDNA genome and recover viable dengue virus mutants that are restricted in viral replication. DEN4 deletion mutants should have the advantage of being less subject to reversion of phenotype than amino acid substitution mutants. For this reason, a series of DEN4 mutants containing a deletion in the 5' or 3' non-coding (NC) region were constructed. Growth analysis indicated that most of these DEN4 deletion mutants exhibited reduced replicative capacity in simian cell culture. Experience with several well-studied live vaccines directed against other viruses indicates that reduced viral replication in cell culture is usually associated with attenuation for humans. Earlier, several DEN4 3? NC deletion mutants that exhibited reduced growth efficiency in simian LLC-MK2 cells were selected for evaluation of their infectivity and immunogenicity in rhesus monkeys. The results of this study indicated that occurrence and duration of viremia were reduced for some of the deletion mutants compared to the parental wild type virus. Hence, we felt that one or more DEN4 3? deletion mutants might prove to be useful for immunization of humans against disease caused by dengue virus. Simian Vero cells and cells of a primary African green monkey kidney (AGMK) line were recently certified for production of human vaccines. We previously showed that DEN4 replicated efficiently in both Vero and AGMK cells. For this reason, cDNA- derived DEN4 originally recovered from transfected mosquito C6/36 cells was subjected to further passage in Vero cells and primary AGMK cells (kindly supplied by Dr. Lou Potash, DynCorp Inc., Rockville, MD). These DEN4 preparations were used to inoculate monkeys to determine their immunogenicity in comparison with DEN4 recovered from simian LLC-MK2 cells or mosquito C6/36 cells. Immunization with DEN4 prepared in Vero cells or in AGMK cells induced a high titer of serum neutralizing antibodies, similar to the level detected in monkeys inoculated with DEN4 recovered from mosquito C6/36 cells or simian LLC-MK2 cells. The simian Vero cell line was selected for growth of DEN4 candidate vaccines to be studied in future clinical trials. These clinical vaccine studies will be performed in collaboration with Drs. Brian Murphy and Robert Chanock (LID, NIAID), and Drs. MaryLou Clements and Donald Burke (Johns Hopkins University). The first candidate DEN4 vaccine, mutant 3?d 172-143, was selected on basis of its growth restriction in simian cells in vitro as well as monkeys in vivo. This mutant was passaged three times in Vero cells in serum-free medium to prepare a pre-vaccine pool from which the final vaccine lot was produced. During each passage, there was an increase in the virus yield, reaching a titer of <107 pfu/ml in the vaccine lot. Further concentration and purification of virus was not necessary as the level of cell DNA in the preparation was sufficiently low enough to satisfy FDA requirements. The complete nucleotide sequence of the genome of the DEN4 vaccine was also determined to confirm the presence of the 3? NC deletion and the absence of any new adventitous mutations. Extensive safety tests have been nearly completed for the bulk vaccine preparation as well as the final vaccine container materials. Following successful completion of these tests that are required for FDA approval, we will file an IND for clinical trials in healthy adults.